Association of IL-4 with pachychoroid neovasculopathy

The purpose of this study was to identify the inflammatory cytokines that were associated with pachychoroid neovasculopathy (PNV). Seventy-five eyes of 75 patients with PNV, 145 eyes of 145 patients with neovascular age-related macular degeneration without pachyvessels, and 150 eyes of 150 normal subjects were examined for the levels of intraocular cytokines. In eyes with PNV, the levels of IL-1α, IL-1β, IL-2, IL-4, IL-10, and VEGF were significantly higher than that of the controls. Logistic regression analysis showed that the highest association with the pachyvessels was found for IL-4, IL-2, and IL-1α. In eyes with PNV, the levels of IL-4, IL-2, IL-5, IL-13, IL-1α, and IL-1β were significantly higher in eyes with both increased choroidal thickness and choroidal vessel diameter. The strongest correlation with the choroidal thickness and vessel diameter was observed for IL-4. In PNV eyes with polypoidal lesions, the levels of IL-4, IL-17, and TNFβ were significantly correlated with the number of polypoidal lesions. Of these cytokines, IL-4 was especially associated with the thickness of the choroidal vessels and the formation of polypoidal lesions. We conclude that IL-4 is most likely involved in establishing the clinical characteristics of PNV and polypoidal vascular remodeling.


Results
Association of inflammatory cytokines with pachychoroid neovasculopathy (PNV) and with neovascular age-related macular degeneration (nAMD) without pachyvessels. The levels of the intracameral cytokines were determined in 75 eyes of 75 patients with PNV, 145 eyes of 145 patients with nAMD without pachyvessels including 93 eyes of 93 patients with polypoidal choroidal vasculopathy (PCV), and 52 eyes of 52 patients with typical AMD without polypoidal lesions. As control, the levels of intracameral cytokines of 150 eyes of 150 normal subjects who underwent cataract surgery were also determined.
The mean age of the PNV patients was 71.3 ± 1.2 years, of the PCV patients was 74.3 ± 0.9 years, and of the typical AMD patients was 75.7 ± 1.2 years ( Table 1). The mean age of the control subjects were 73.9 ± 0.6 years (all P > 0.05). Women made up 21.3% of the PNV eyes, 30.1% of the PCV eyes, 30.8% of the typical AMD eyes, and 55.3% of the control eyes.
Drusen (non-extensive drusen or hard drusen) 14 was detected in 22.7% of PNV eyes, 16.1% of the PCV eyes, and 26.9% of the typical AMD eyes. Polypoidal lesions were found in 56 eyes of the PNV patients (74.7%).
Because the PNV spectrum of diseases is characterized by a dilation of the choroidal vessels, we first compared the diameter of the choroidal vessels and the thickness of the choroid in the PNV, PCV, and typical AMD eyes. The PNV eyes had significantly thicker choroids and larger choroidal vessels than that of the PCV (P < 0.001) or typical AMD (P < 0.001) eyes (Table 1).
We then assessed the levels of 17 intracameral cytokines in eyes with PNV, PCV, and typical AMD (Fig. 1). In the PNV eyes, the levels of IL-1α, IL-1β, IL-2, IL-4, IL-10, and VEGF were significantly higher than that of the control eyes. In the PCV eyes, the levels of IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, and VEGF were significantly higher than the control eyes, and in the typical AMD eyes, the levels of IL-1α, IL-2, IL-8, IL-10, IL-17, and VEGF were also significantly higher.
These findings suggested that eyes with PNV have overlapping levels of cytokines with eyes with typical AMD and PCV although different levels of elevations were observed.
Thus, in PNV eyes, choroidal thickness, choroidal vessel diameter, and number of polypoidal lesions were associated with elevated levels of inflammatory cytokines.
To calculate direct effect of the inflammatory cytokines on choroidal thickness and choroidal vessel diameter, multivariate linear regression analyses were conducted after adjustments of the number of polypoidal lesions. The results indicated that in eyes with PNV, the elevations of the levels of IL-4, TNFα, IL-17, IL-2, IL-12, IL-15, IL-5, Figure 1. Levels of cytokines in the aqueous humor of eyes with pachychoroid neovasculopathy (PNV), polypoidal choroidal vasculopathy (PCV), typical age-related macular degeneration (typical AMD), and normal subjects. In PNV (red bar), the level of IL-1α, IL-1β, IL-2, IL-4, IL-10, and VEGF were significantly higher than that of the control eyes (white bar). In the PCV eyes (blue bar), IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, and VEGF were significantly higher than that of the control eyes. The levels of IL-1α, IL-2, IL-8, IL-10, IL-17, and VEGF were higher in eyes with typical age-related macular degeneration (AMD; green bar) than in the normal control eyes. PNV, pachychoroid neovasculopathy; PCV, polypoidal choroidal vasculopathy; typical AMD, typical agerelated macular degeneration. Data are the means (pg/ml) and standard error of the means. PNV, n = 75; PCV, n = 93; typical AMD, n = 52; control, n = 150. ANOVA and post hoc test (Dunnett). www.nature.com/scientificreports/ IL-13, IL-1α, and IL-1β were significantly correlated with an increased choroidal thickness (Table 2). Similarly, the levels of IL-4, IL-2, IL-5, IL-10, IL-13, TNFβ, IL-1α, and IL-1β were significantly correlated with larger choroidal vessel diameter (Table 3). Of all these cytokines, IL-4 had the highest contribution with the increase of 11.6 μm in the choroidal thickness and 6.72 μm in the choroidal vessel diameter by its unit increase.

Modeling of association of choroidal thickness, choroidal pathological findings, and inflammatory cytokines. The findings indicated that IL-4 and inflammatory cytokines had a significant effect
on the choroidal thickness and the remodeling of the choroidal vasculature. Therefore, we examined how they contributed to the clinical characteristics including the choroidal thickness, polypoidal lesions, type 1 macular neovascularization (MNV), subretinal fibrosis, and subretinal hemorrhage. We first analyzed the associations of all the clinical characteristics using a structural equation modeling. After the construction of a model of the clinical characteristics (Fig. 2), the involvement of the significantly elevated cytokines was assessed. We found that IL-4 showed the highest significant association with the development of the clinical characteristics including the choroidal thickness, and polypoidal lesions. A path diagram indicated that the IL-4/quintile significantly increased the choroidal thickness by 0.28 quintile (P < 0.001) as well as the vessel diameter by 0.30 quintile (P < 0.001) (Supplementary Figure S1). The effects of IL-4 on the number of polypoidal lesions was almost one-half of that on the choroidal thickness.
Interestingly, IL-4 had no direct association with the presence of subretinal hemorrhages or subretinal fibrosis. The number of polypoidal lesions/quintile was significantly associated with the presence of subretinal hemorrhages (P < 0.001).

Discussion
The pachychoroid spectrum of diseases including PNV is a recently proposed disease entity characterized by pachyvessels 1,3 . Our results showed that IL-4 was associated with the presence of pachyvessels in the choroid and with the PNV spectrum of diseases. More specifically, the remodeling of the pachyvessels was associated with higher levels of IL-4 expression, which was partly associated with the formation of the polypoidal lesions. These findings suggested that IL-4 was associated with the vascular remodeling of the choroidal vessels and may play different roles in the PNV disease spectrum.
In the PNV disease spectrum, IL-4 was specifically associated with the choroidal thickness and blood vessel diameter, and both were associated with a single nucleotide polymorphism, IL-4 -590 (rs2243250). This nucleotide is a well-recognized allele associated with asthma 15 and rhinitis 16 , and its presence leads to an overexpression of the mRNA of IL-4 17 and increased levels of plasma IL-4 18 .
In contrast, there have been few reports on the associations of PNV. Earlier studies showed that PNV was associated with elevated levels of VEGF 6 , CCL2 (C-C motif chemokine 2) 6,25 , IL-8, angiopoietin-2, PGF (placental growth factor), CXCL12 (C-X-C Motif Chemokine Ligand 12), and CXCL13 (C-X-C Motif Chemokine Ligand 13) 25 . Thus, eyes with PNV have an overlapping profile of cytokine elevations with nAMD, however it is still unclear whether PNV has a specific cytokine profile.
PNV is characterized by the presence of pachyvessels, vascular hyperpermeability, and vasodilation of the outer choroidal vessels. Thus, the vascular hyperpermeability of the pachyvessels causes choroidal thickening and subretinal exudative changes 10 . However, previous reports on PNV assessed its association with the disease category, and no detailed analyses for the disease characteristics have been published. Thus, it remains unclear how the observed cytokines may be associated with or contribute to the formation of pachyvessels or vascular remodeling.
The critical disease-associated cytokines for PNV appeared to be VEGF based on the previous information on nAMD. However, in PNV, the contribution of the VEGF in the aqueous to PNV is inconsistent depending on the study. Kato et al. reported an elevation of VEGF in PNV eyes 6 , however the most recent study revealed that VEGF was significantly decreased in the eyes without drusen or decreased in eyes with non-pachychoroid, non-drusen, and it is likely that the presence of drusen can be associate with the VEGF level 25 . The same report showed that an elevation of CRP, CXCL12, CXCL13, IL-8, angiopoietin-2, PIGF, and CCL2 was present in eyes www.nature.com/scientificreports/ with PNV 25 . Although the involvement of various types of drusen on cytokine kinetics has not been fully analyzed, an elevation of IL-8 and CCL2 was not observed in PNV eyes in our cohort. Because CCL2 is well recognized to be associated with nAMD, we also examined the association of CCL2 with choroidal thickness by structural equation modeling analyses. We found a significant and negative effect of CCL2 on the choroidal thickness (coefficient: -0.18, P = 0.007). However, the inclusion of CCL2 into the structural equation modeling analysis did not significantly change the structure of the outcomes. Thus, the role of CCL2 has not been determined and presumably may be context dependent.
T-helper 2 (Th2) lymphocytes and related cytokines including IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25) play significant roles in the increased vascular permeability and vasodilatability. Of these, IL-4 decreases the endothelial barrier function which causes the vascular hyperpermeability 26,27 . Importantly, the IL-4-mediated hyperpermeability of the vascular endothelial cells has been shown to be a major mechanism for inflammatory edema 28 .
Mechanistically, IL-4 impairs the barrier function of the vascular endothelium by forming gaps between the endothelial cells through the rearrangement of the cytoskeleton 29 . Another mechanism for the IL-4 mediated barrier dysfunction has been recently reported. IL-4 stimulates the formation of actin stress fibers by a noncanonical Wnt ligand, Wnt5A 30 . This eventually causes a remodeling of the cytoskeleton leading to an impairment of the barrier function 30 . Thus, we propose that inflammatory networks featuring IL-4 contributes to vascular hyperpermeability leading to the development of the pachyvessels.
Another characteristic of pachyvessels is the vasodilation of the outer choroidal vessels. Vasodilation has also been reported to be associated with IL-4. IL-4 promotes both the lumen formation 13,31 and the endothelial cell cycle 32 . In addition, IL-4 stimulates the VCAM-1 promoter via STAT6 33 and exerts proangiogenic effects on the VCAM-1/alpha4 integrin pathway 31 . In addition, the vasodilation itself induces chronic ischemia and the hypoxic condition of the choriocapillaris 2 leads to the induction of IL-4 34 .
Considering the pathogenic mechanism of PNV to be a venous overload choroidopathy, PNV is similar to diseases with chronic venous congestions 3 . A wide variety of venous disorders throughout the body including varicose veins 35 , pregnancy 36,37 , pelvic congestion syndrome 38 , and gastrointestinal varices associated with cirrhosis and portal hypertension 39 are characterized by vasculopathies due to venous overload. The venous overload promotes capillary dilation and increases the permeability, inflammatory cell infiltration, activation of matrix metalloproteinases (MMPs) 40 , and the induction of cell adhesion molecules (CAMs) 41 .
Importantly, IL-4 has been shown to be associated with the development of vasodilatability, hypervascular permeability, and tissue vascular remodeling which is exemplified in the placenta during pregnancy 42 and the regeneration of cardiomyocytes 43 .
Analysis of the aqueous humor showed that IL-4 was also associated with the polypoidal lesions 11 . In addition, elevated IL-4 levels may induce polypoidal formation or choroidal neovascularizations 13 . Polypoidal lesions are often accompanied by pachyvessels 44 . Because large polypoidal lesions can be regarded as neovascular tangles or branching neovascular networks rather than actual polypoidal lesions or aneurysmal dilatations in OCT images 45 , polypoidal lesions may reflect angiogenic lesions 44,46 . Our results showed that IL-4 was significantly associated with the choroidal thickness and the presence of polypoidal lesions in eyes with PNV. This is consistent with reports that showed that the development of polypoidal lesions was closely associated with development of pachyvessels 47 . Collectively, the PNV diseases appeared to be associated with vascular remodeling including the development of polypoidal lesions. In addition, the pachyvessels may also promote choroidal neovascularizations 48 .
There are some limitations in this study. Our study was a retrospective analysis and may not have a direct causal relationship. For example, there is a possibility that the contribution of IL-4 may be indirect effects of unobserved factors or molecules. However, our previous analyses using IL-4 deficient mice supported the significant and direct roles of IL-4 in vascular remodeling which supports our conclusions 13 . To understand the roles of IL-4 in greater detail, clinical trials of blocking IL-4 in PNV patients are required. Nevertheless, our study provides a strong basis for future therapy blocking the IL-4 pathway to treat patients with PNV spectrum of diseases.
In conclusion, our findings indicate that IL-4 represents a crucial mediator in the development of the PNV spectrum of diseases as well as the vascular remodeling in PNV patients.

Methods
Diagnosis and eligibility criteria of patients. All of the consecutive cases of PNV and nAMD that received intravitreal ranibizumab or aflibercept at the University of Tottori Hospital between 2013 January and 2018 September were studied.
Those who met the criteria for PNV were designated as PNV and the others as nAMD. We used the following criteria for the diagnosis of PNV which was based on earlier reports 1,8,49,50 : (1) presence of type 1 macular neovascularization (MNV) detected in either eye; (2) choroidal vascular hyperpermeability detected in the late phase of indocyanine green angiography (ICGA); (3) presence of dilated choroidal vessels below type 1 MNV detected by optical coherence tomography (OCT); (4) no or only nonextensive drusen or hard drusen in both eyes (The Age-Related Eye Disease Study (AREDS) Report Number 6: Level 1) 14 .
A representative case of typical PNV is shown in Fig. 3. This was a 62-year-old female patient with polypoidal lesions who was diagnosed as having PNV. A retinal pigment epithelium (RPE) abnormality and leaky large choroidal vessels were observed in the ICGA.
PCV was diagnosed in the presence of a branching vascular network with terminal aneurysmal dilatations in the ICGA images corresponding to an RPE elevation in the OCT images 44 .
Typical AMD was diagnosed by the presence of MNV and other findings corresponding to the AREDS Report Number 6, Levels 2, 3, and 4 8,14 (Supplementary Figure S2).   www.nature.com/scientificreports/ The exclusion criteria were eyes with retinal angiomatous proliferation (RAP), MNVs secondary to high myopia with a refractive error (spherical equivalent) ≤ − 6.00 D, trauma, angioid streaks, uveitis, any other neovascular maculopathy, prior laser photocoagulation, photodynamic therapy, and intraocular surgery within the past 3 months.
All the patients underwent a unified diagnosis by three independent examiners (A.K., R.U., and H.M.). If the three diagnoses did not match, the senior investigators (K.I. and S.S.) made the final decision after discussions with other retina specialists.
The IL-4 polymorphism was examined in 14 PNV and 39 nAMD patients without pachyvessels. All procedures were performed with consent from the patient. For the control groups, aqueous humor was collected from 150 eyes of 150 normal patients who underwent routine cataract surgery.
The procedures used in this study were approved by the institutional Review Board of the University of Tottori, and they conformed to the tenets of the Declaration of Helsinki. A signed written informed consent was obtained from all participants.

Measurement of cytokines in aqueous humor.
On the day of the initial anti-vascular endothelial growth factor (anti-VEGF) treatment, 100 μL of aqueous humor was collected by paracentesis before the injection of the anti-VEGF agent and stored at − 80 °C until cytokine measurements.
Sequencing was performed with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Waltham, MA) and a 3500xL Genetic Analyzer capillary sequencer (Thermo Fisher Scientific Inc.) as described 52 . All of the primer sequences are available on request.
The subfoveal choroidal thickness was measured from the retinal pigment epithelium to the choroid-sclera interface at the foveal center. The choroidal vessel diameter (vertical diameter of the thickest outer choroidal vessel in the foveal region) was measured by referring to the scale bars in the OCT system. Statistical analyses. The decimal best-corrected visual acuity (BCVA) was measured with a Landolt chart and converted to the logarithm of minimum angle of resolution (logMAR) for the statistical analyses. Logistic regression analyses were performed to calculate the odds ratios (OR) based on quintiles of each cytokine level. Each cytokine quintile was compared with the lowest quintile as the reference category. Unpaired t tests, Mann-Whitney U tests, and ANOVA with post hoc analysis were used to determine whether the differences between groups were statistically significant. Data are presented as means and standard error of the means.
For the logistic regression analyses and structural equation modeling, the clinical characteristics were coded as (0/1). The model was selected based on fit index (Akaike information criteria (AIC) and Bayesian information criteria (BIC)) 53 .
The statistical analyses were performed using Stata 16.0 (StataCorp, College Station, TX). A P-value < 0.05 was considered statistically significant.

Data availability
The known genetic polymorphism (rs2243250) analyzed during the current study is available in the dbSNP repository, [https:// www. ncbi. nlm. nih. gov/ snp/ rs224 3250]. The other datasets for the current study are available from the corresponding author on reasonable request.